MCH4 and MCH5, apoptotic proteases

ABSTRACT

The invention provides an isolated gene encoding Mch4 or an isolated gene encoding Mch5 as well as functional fragments thereof. Also provided are isolated nucleic acid sequences encoding Mch4 or Mch5 or functional fragment thereof. The gene or nucleic acid sequences can be single or double stranded nucleic acids corresponding to coding or non-coding strands of the Mch4 or Mch5 nucleotide sequences. Isolated Mch4 or Mch5 polypeptides or functional fragments thereof are also provided.

This invention was made with government support under grants AI 35035-01from the National Institutes of Health. Accordingly, the government hascertain rights to this invention.

Throughout this application various publications are referenced withinparentheses. The disclosures of these publications in their entiretiesare hereby incorporated by reference in this application in order tomore fully describe the state of the art to which this inventionpertains.

BACKGROUND OF THE INVENTION

The present invention relates generally to apoptosis or, programed celldeath, and more particularly, to novel aspartate-specific cysteineproteases which can be used to modulate apoptosis for the therapeutictreatment of human diseases.

Apoptosis is a normal physiological process of cell death that plays acritical role in the regulation of tissue homeostasis by ensuring thatthe rate of new cell accumulation produced by cell division is offset bya commensurate rate of cell loss due to death. It has now become clearthat disturbances in apoptosis, also referred to as physiological celldeath or programmed cell death, that prevent or delay normal cellturnover can be just as important to the pathogenesis of diseases as areknown abnormalities in the regulation of proliferation and the cellcycle. Like cell division, which is controlled through complexinteractions between cell cycle regulatory proteins, apoptosis issimilarly regulated under normal circumstances by the interaction ofgene products that either induce or inhibit cell death.

The stimuli which regulate the function of these apoptotic gene productsinclude both extracellular and intracellular signals. Either thepresence or the removal of a particular stimuli can be sufficient toevoke a positive or negative apoptotic signal. For example,physiological stimuli that prevent or inhibit apoptosis include, forexample, growth factors, extracellular matrix, CD40 ligand, viral geneproducts neutral amino acids, zinc, estrogen and androgens. In contrast,stimuli which promote apoptosis include growth factors such as tumornecrosis factor (TNF), Fas, and transforming growth factor β (TGFβ),neurotransmitters, growth factor withdrawal, loss of extracellularmatrix attachment, intracellular calcium and glucocorticoids, forexample. Other stimuli, including those of environmental andpathogenetic origins, also exist which can either induce or inhibitprogrammed cell death. Although apoptosis is mediated by diverse signalsand complex interactions of cellular gene products, the results of theseinteractions ultimately feed into a cell death pathway that isevolutionarily conserved between humans and invertebrates.

Several gene products which modulate the apoptotic process have now beenidentified. Although these products can in general be separated into twobasic categories, gene products from each category can function toeither inhibit or induce programmed cell death. One family of geneproducts are those which are members of the Bcl-2 family of proteins.Bcl-2, is the best characterized member of this family and inhibitsapoptosis when overexpressed in cells. Other members of this gene familyinclude, for example, Bax, Bak, Bcl-x_(L), Bcl-x_(S), and Bad, Whilesome of these proteins can prevent apoptosis others augment apoptosis(e.g. Bcl-x_(S) and Bak, respectively).

A second family of gene products, the aspartate-specific cysteineproteases (ASCPs), are related genetically to the C. elegans ced-3 geneproduct which was initially shown to be required for programmed celldeath in the roundworm, C. elegans. The ASCPs family of proteasesincludes human ICE (interleukin-1-β converting enzyme), ICH-1_(L),ICH-1_(S), CPP32, Mch2, Mch3, ICH-2 and ICE_(rel) ⁻ III. Among thecommon features of these gene products is that 1) they are cysteineproteases with specificity for substrate cleavage at Asp-x bonds, 2)they share a conserved pentapeptide sequence (QACRG) within the activesite and 3) they are synthesized as proenzymes that require proteolyticcleavage at specific aspartate residues for activation of proteaseactivity. Cleavage of the proenzyme produces two polypeptide proteasesubunits of approximately 20 kD (p20) and 10 kD (p10) which, in the caseof ICE, combine non-covalently to form a tetramer comprised of twop20:p10 heterodimers. Although these proteases, when expressed in cells,induce cell death, several alternative structural forms of theseproteases, such as ICESδ, ICEε, ICH-1_(S) and Mch2β, actually functionto inhibit apoptosis.

In addition to the Bcl-2 and ASCP gene families which play a role inapoptosis in mammalian cells, it has become increasingly apparent thatother gene products exist which are important in mammalian cell deathand which have yet to be identified. For example, in addition to Ced-3,another C. elegans gene known as Ced-4 exists which is also required forprogrammed cell death in C. elegans. However, mammalian homologues ofthis protein remain elusive and have not yet been identified. Further,it is ambiguous as to whether other genes exist which belong to eitherof the above two apoptotic gene families or what role they may play inthe programmed cell death pathway. Finally, it is unclear what thephysiological control mechanisms are which regulate programmed celldeath or how the cell death pathways interact with other physiologicalprocesses within the organism. For example, recently it has beensuggested that cytotoxic T-lymphocytes mediate their destructivefunction by inducing apoptosis in their target cells.

Apoptosis functions in maintaining tissue homeostasis in a range ofphysiological processes such as embryonic development, immune cellregulation and normal cellular turnover. Therefore, the dysfunction, orloss of regulated apoptosis can lead to a variety of pathologicaldisease states. For example, the loss of apoptosis can lead to thepathological accumulation of self-reactive lymphocytes such as thatoccurring with many autoimmune diseases. Inappropriate loss of apoptosiscan also lead to the accumulation of virally infected cells and ofhyperproliferative cells such as neoplastic or tumor cells. Similarly,the inappropriate activation of apoptosis can also contribute to avariety of pathological disease states including, for example, acquiredimmunodeficiency syndrome (AIDS), neurodegenerative diseases andischemic injury. Treatments which are specifically designed to modulatethe apoptotic pathways in these and other pathological conditions canchange the natural progression of many of these diseases.

Thus, there exists a need to identify new apoptotic genes and their geneproducts and for methods of modulating this process for the therapeutictreatment of human diseases. The present invention satisfies this needand provides related advantages as well.

SUMMARY OF THE INVENTION

The invention provides an isolated gene encoding Mch4 or an isolatedgene encoding Mch5 as well as functional fragments thereof. Alsoprovided are isolated nucleic acid sequences encoding Mch4 or Mch5 orfunctional fragments thereof. The gene or nucleic acid sequences can besingle or double stranded nucleic acids corresponding to coding ornon-coding strands of the Mch4 or Mch5 nucleotide sequences. IsolatedMch4 or Mch5 polypeptides or functional fragments thereof are alsoprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide and predicted amino acid sequence of Mch4(SEQ ID NOS:1 and 2, respectively).

FIG. 2 shows the nucleotide and predicted amino acid sequence of Mch5(SEQ ID NOS:3 and 4, respectively).

FIG. 3 shows the predicted amino acid sequences of Mch4 and Mch5 andtheir homology to other ASCP sequences. (A) Colinear alignment of Mch4and Mch5 Proteins. Dotted lines indicated gaps in the sequence to allowoptimal alignment. Amino acid residues are numbered to the right of eachsequence. (B) Multiple sequence alignment of all known human ASCPs andthe nematode Ced-3 ASCP. The active site pentapeptide QACRG/QACQG (SEQID NOS:11 ang 10, respectively) is boxed. Based on crystal structure ofICE, the numbered residues within the ICE sequence are involved incatalysis (open boxes), and binding the substrate-carboxylate of P1 Asp(open circles). The residues adjacent to the substrate P2-P4 amino acidsare indicated by closed triangles. D/X indicates known and potentialprocessing sites between the small and large subunits of ASCPs. TheRoman numbers on the right indicate the three ASCP-subfamilies; theCed-like subfamily (I), the ICE-like subfamily (II) and the Nedd2/Ich-1subfamily (III). The asterisk indicates the nonconservative Arg to Glnsubstitution in Mch4 and Mch5.

FIG. 4 shows the cleavage of CPP32 proenzyme by Mch4 and granzyme B. (A)Effect of Asp175 mutation on cleavage of proCPP32. ³⁵ S-labeled wildtype proCPP32 (Mut-D175, -lanes) or AsP175-mutated proCPP32 (Mut-D175,+lanes) were incubated with recombinant Mch4 (Mch4, +lanes), granzyme B(GraB, +lanes) or buffer (Mch4 and GraB, -lanes) for 1 h at 37° C. Thereaction products were then analzyed by SDS-PAGE and autoradiography.(B) Effect of Asp9 mutation and the DEVD-CHO inhibitor on cleavage ofthe propeptide of proCPP32. 35S-labeled wild type proCPP32 (mut-D9 andMut-175, -lanes) or Asp9-mutated (mut-D9, +lanes) or Asp 175-mutated(Mut-D175, +lanes) proCPP32 were incubated with granzyme B (GraB,+lines) or buffer (GraB, -lanes) in the presence (+lanes) or absence(-lanes) of the DEVD-CHO inhibitor.

The reaction products were analyzed as above. SS, indicates the smallsubunit. LS, indicates the large subunit.

FIG. 5 shows Cleavage of Mch3 and Mch4 proenzymes by Mch4 and granzymeB. (A) Effect of Asp198 mutation on cleavage of proMch3. ³⁵ S-labeledwild type proMch3 (Mut, -lanes) or Asp198-mutated proMch3 (Mut, +lanes)were incubated with recombinant Mch4 (Mch4, +lanes), granzyme B (GraB,+lanes) or buffer (Mch4 and GraB, -lanes) for 1 h at 37° C. The reactionproducts were then analyzed by SDS-PAGE and autoradiography. The firstthree lanes contain truncated proMch4 (amino acids 102-346) treatedexactly as above. SS, indicates the small subunit. LS, indicates thelarge subunit.

FIG. 6 shows CPP32 and Mch3 activity towards Mch4 proenzyme. 35S-labeledwild type proMch4 (Mut, -lanes) or Asp239-mutated proMch4 (Mut, +lanes)were incubated with recombinant CPP32 (CPP32, +lanes), Mch3 (Mch3,+lanes) or buffer (CPP32 and Mch3, -lanes) for 1 h at 37° C. Thereaction products were then analyzed by SDS-PAGE and autoradiography.

FIG. 7 shows potential apoptotic protease cascades.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to novel cell death specific proteases termedMch4 and Mch5. These proteases are members of the aspartate-specificcysteine protease (ASCP) family of proteases which includes, forexample, ICE, ICH-1_(L), ICH-1_(S), CPP32, Mch2, Mch3, ICH-2 andICE_(rel) ⁻ III. Similar to other ASCPS, Mch4 and Mch5 are synthesizedas a larger proenzyme and become active following proteolytic cleavageinto two subunits; large subunit of approximately 17-27 kD and smallsubunit of approximately 10-12 kD. The two subunits form heterodimerswhich associate with each other into an active complex. Substratespecificity uniquely requires an Asp residue in the P1 position of thesubstrate binding site with a small, preferably hydrophobic, residue inthe P1' position.

In one embodiment, the invention is directed to nucleic acids encodingthe apoptotic cysteine protease Mch4 or Mch5. The nucleic acids are usedto produce recombinant Mch4 or Mch5 proteases, whose activity can bemeasured enzymatically. The recombinant polypeptides are used to screenfor Mch4 or Mch5 inhibitory compounds. Such pharmaceutical compounds areuseful for the treatment or prevention of diseases which arecharacterized by apoptotic cell death. Alternatively, the Mch4 or Mch5polypeptides can be used to screen for pharmaceutical compounds whichactivate or act as agonists of Mch4 or Mch5 such as by inducing cleavageof the proenzyme into its active subunits. Such compounds are useful forthe treatment or prevention of diseases which are characterized by theloss of apoptotic cell death.

As used herein, the term "substantially" when referring to a Mch4 orMch5 nucleotide or amino acid sequence is intended to refer to thedegree to which two sequences of between about 15-30 or more nucelotidesin length, are identical or similar so as to be considered by thoseskilled in the art to be functionally equivalent. For example, the Mch4or Mch5 nucleic acids of the invention have a nucleotide sequencesubstantially the same as that shown in FIGS. 1 and 2 and as SEQ IDNOS:1 and 3, respectively. Thus, if a second sequence is substantiallythe same as that shown as SEQ ID NOS:1 and 3, then it is consideredfunctionally equivalent by those skilled in the art. Methods forsequence comparisons and determinations of similarity are well known androutine within the art.

Functionally equivalent nucleic acid sequences include, for example,sequences that are related, but different and encode the same Mch4 orMch5 polypeptide due to the degeneracy of the genetic code as well assequences that are related, but different and encode a different Mch4 orMch5 polypeptide that exhibits similar functional activity. In bothcases, the nucleic acids encode functionally equivalent gene products.Functional fragments of Mch4 or Mch5 encoding nucleic acids such asoligonucleotides, polyoligonucleotides, primers and the like are alsoconsidered to be within the definition of the term and the invention asclaimed. Functional equivalency is also relevant to Mch4 or Mch5 nucleicacids which do not encode gene products, for example, but instead arefunctional elements in and of themselves. Specific examples of suchfunctional nucleic acids include, for example, promoters, enhancers andother gene expression regulatory elements.

Mch4 or Mch5 polypeptides of the invention have an amino acid sequencesubstantially similar to that shown in FIGS. 1, 2 and 3 and in SEQ IDNOS:2 and 4, respectively. Functionally equivalent Mch4 amino acidsequences similarly includes, for example, related, but differentsequences so long as the different polypeptide exhibits at least onefunctional activity of Mch4 or Mch5. Such related, but differentpolypeptides include, for example, substitutions of conserved andnon-essential amino acids. Fragments and functional domains of Mch4 orMch5 are similarly included within the definition of the term and theclaimed invention.

Therefore, it is understood that limited modifications may be madewithout destroying the biological function of the Mch4 or Mch5polypeptide and that only a portion of the entire primary structure maybe required in order to effect activity. For example, minormodifications of the Mch4 or Mch5 amino acid sequences (SEQ ID NOS:2 and4) which do not destroy their activity also fall within the definitionof Mch4 or Mch5 and within the definition of the polypeptide claimed assuch. Also, for example, genetically engineered fragments of Mch4 orMch5 either alone or fused to heterologous proteins such as fusionproteins that retain measurable enzymatic or other biological activityfall within the definition of the polypeptides claimed as such.

It is understood that minor modifications of primary amino acid sequencemay result in polypeptides which have substantially equivalent orenhanced function as compared to the sequences set forth in FIGS. 1 and2 (SEQ ID NOS:2 and 4). These modifications may be deliberate, asthrough site-directed mutagenesis, or may be accidental such as throughmutation in hosts which are Mch4 or Mch5 producers. All of thesemodifications are included as long as Mch4 or Mch5 biological functionis retained. Further, various molecules can be attached to Mch4 or Mch5,for example, other proteins, carbohydrates, lipids, or chemicalmoieties. Such modifications are included within the definition of Mch4or Mch5 polypeptides.

The invention provides a gene encoding Mch4 or Mch5, or fragmentthereof. The invention also provides an isolated nucleic acid sequenceencoding Mch4 or Mch5, or fragment thereof. The gene and nucleic acidsequences encode substantially the sequence as shown in SEQ ID NOS:1 and3. Fragments of the gene or nucleic acid sequence are provided whichcomprise single or double stranded nucleic acids having substantiallythe sequences shown in SEQ ID NOS:1 and 3.

The Mch4 or Mch5 nucleic acids of the present invention were identifiedand isolated by a novel approach of searching a human database ofexpressed sequence tags (ESTs) under various stringencies to identifypotential new sequence fragments which may have homology to the ICEfamily of cysteine proteases. As described below, such a searchidentified the Mch4 and Mch5 nucleic acids of the present invention andalso resulted in the reclassification of the cell death protease family.Previously these proteaes were referred to as the ICE-family ofproteases and thus the initial search criteria was directed to "ICEfamily" of cell death proteases. However, with the identification ofMch4 and Mch5, the proteases can now be divided into three subfamiliesreferred to herein as the Ced-like, ICE-like and Nedd2/ICH-1-likesubfamilies of cell death proteases (see FIG. 3B).

In regard to the search for potential new sequences having homology tothe previously referred to ICE family of proteases, novel sequencesidentified from the search as having homology to the ICE family of celldeath proteases are then used to design primers for attempting PCRamplification and cloning of the actual cDNA. The second primer for theamplification is designed to encompass homologous regions in nucleicacid sequences that encode known ICE protease family members. In thisspecific case, the primer was directed to the GSWFI/GSWYI (SEQ ID NOS:62and 63, respectively) pentapeptide sequence that is conserved in anumber of the ICE/Ced-3 family of proteases. The primer design shouldtake into account the predicted strandedness of both the EST sequenceprimer and the known primer. Thus, only if the homology search andprimer hybridization conditions are successfully determined, will suchan approach allow PCR amplification of a fragment of the putative novelprotease cDNA.

As searching a genetic data base will yield homologous sequence matchesto any query nucleotide sequence, additional criteria must be used toidentify the authentic ICE subfamily homologue from among thenonspecific homology matches. ICE family members share the highestdegree of homology in the active site and catalytically important aminoacid residues. A given EST returned by the search may not include one ofthese highly homologous sites, but rather, may only include a regionwithin the protease with cryptic homology. Confirming an EST as a novelICE protease involves translation of all the positive EST hits in threedifferent reading frames and subsequent identification of conservativeactive site or catalytically important amino acid sequence motifs. Then,using conventional cDNA cloning, a full length cDNA of the putativenovel protease can be obtained and 1) analyzed for overall structuralhomology to ICE family members, 2) recombinantly expressed and analyzedfor cysteine protease activity, and 3) analyzed for the induction ofprogrammed cell death by heterologous expression of the cDNA inappropriate cells.

Alternative methods than that described above for isolating Mch4 or Mch5encoding nucleic acids can similarly be employed. For example, using theteachings described herein, those skilled in the art can routinelyisolate and manipulate Mch4 or Mch5 nucleic acids using methods wellknown in the art. All that is necessary is the sequence of the Mch4 orMch5 encoding nucleic acids (FIGS. 1 and 2 and SEQ ID NOS:1 and 3) ortheir amino acid sequences (FIGS. 1 and 2 and SEQ ID NOS:2 and 4).

Such methods include, for example, screening a cDNA or genomic libraryby using synthetic oligonucleotides, nucleic acid fragments or primersas hybridization probes. Alternatively, antibodies to the Mch4 or Mch5amino acid sequence or fragments thereof can be generated and used toscreen an expression library to isolate Mch4 or Mch5 encoding nucleicacids. Other binding reagents to Mch4 or Mch5 polypeptides can similarlybe used to isolate Mch4 or Mch5 polypeptides having substantially theamino acid sequence show in FIGS. 1 and 2. Similarly, substrate reagentssuch as non-cleavable peptide analogues of cysteine proteases can beused to screen and isolate Mch4 or Mch5 polypeptides.

In addition, recombinant DNA methods currently used by those skilled inthe art include the polymerase chain reaction (PCR) which, combined withthe Mch4 or Mch5 nucleotide and amino acid sequences described herein,allows reproduction of Mch4 or Mch5 encoding sequences. Desiredsequences can be amplified exponentially starting from as little as asingle gene copy by means of PCR. The PCR technology is the subjectmatter of U.S. Pat. Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202all of which are incorporated by reference herein.

The above described methods are known to those skilled in the art andare described, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1992) andthe various references cited therein and in Ansubel et al., CurrentProtocols in Molecular Biolocry, John Wiley and Sons, Baltimore, Md.(1989); and in Harlow et al., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York (1989). These references and thepublications cited therein are hereby expressly incorporated herein byreference.

The invention provides an isolated Mch4 or Mch5 polypeptide comprisingsubstantially the amino acid sequence as that shown in FIGS. 1 and 2(SEQ ID NOS:2 and 4). Mch4 or Mch5 functional fragments are alsoprovided. A specific example of an Mch4 or Mch5 functional fragment isthe catalytic domain which contains the active site amino acid sequenceQACQG. When compared to the active site amino acid sequence of otherASCP family members, QACRG, this active site sequence is similar butdiffers at position 4 with R substituted by Q.

Isolated Mch4 or Mch5 polypeptides of the invention can be obtained by avariety of methods known within the art. For example, the isolatedpeptides can be purified by biochemical methods including, for example,affinity chromatography. Affinity matrices which can be used for Mch4 orMch5 isolation can be anti-Mch4 or anti-Mch5 monoclonal or polyclonalantibodies prepared against the sequence shown in FIGS. 1 and 2 (SEQ IDNOS:2 and 4), or fragments thereof such as synthetic peptides.Alternatively, substrate analogues or enzymatic inhibitors of Mch4 orMch5 can similarly be used as affinity matrices to isolate substantiallypure Mch4 or Mch5 polypeptides of the invention.

Mch4 or Mch5 polypeptides can also be produced by recombinant methodsknown to those skilled in the art. Recombinant Mch4 or Mch5 polypeptidesinclude, for example, an amino acid sequence substantially the same asthat shown in FIGS. 1 and 2 (SEQ ID NOS:2 and 4) as well as fusionproteins and fragments thereof. The Mch4 or Mch5 encoding nucleic acidscan be cloned into the appropriate vectors for propagation, manipulationand expression. Such vectors are known or can be constructed by thoseskilled in the art and should contain all expression elements necessaryfor the transcription, translation, regulation, and if desired, sortingof the Mch4 or Mch5 polypeptides. The vectors can also be for use ineither procaryotic or eucaryotic host systems so long as the expressionand regulatory elements are of compatible origin. One of ordinary skillin the art will know which host systems are compatible with a particularvector. The recombinant polypeptides produced can be isolated by themethods described above.

Apoptosis plays a significant role in numerous pathological conditionsin that programed cell death is either inhibited, resulting in increasedcell survival, or enhanced which results in the loss of cell viability.Examples of pathological conditions resulting from increased cellsurvival include cancers such as lymphomas, carcinomas and hormonedependent tumors. Such hormone dependent tumors include, for example,breast, prostrate and ovarian cancer. Autoimmune diseases such assystemic lupus erythematosus and immune-mediated glomerulonephritis aswell as viral infections such as herpesvirus, poxvirus and adenovirusalso result from increased cell survival or the inhibition of apoptosis.

In contrast, apoptotic diseases where enhanced programed cell death is aprevalent cause generally includes, for example, degenerative disorderssuch as Alzheimer's disease, Parkinson's disease, Amyotrophic lateralsclerosis, Retinitis pigmentosa, and Cerebellar degeneration. Otherdiseases associated with increased apoptosis include, for example,myelodysplastic syndromes such as aplastic anemia and ischemic injuryincluding myocardial infarction, stroke and reperfusion injury.

The Mch4 or Mch5 encoding nucleic acids and polypeptides of theinvention can be used to diagnose, treat or reduce the severity of celldeath mediated diseases such as those described above as well as otherdiseases mediated by either increased or decreased programmed celldeath. Additionally, the Mch4 or Mch5 encoding nucleic acids andpolypeptides of the invention can be used to screen for pharmaceuticalcompounds and macromolecules which inhibit or promote Mch4 mediatedapoptosis.

For example, the Mch4 or Mch5 encoding nucleic acids, polypeptides andfunctional fragments thereof can be used to diagnose, or to generatereagents to diagnose diseases mediated or characterized by programedcell death. Diagnosis can be by nucleic acid probe hybridization withMch4 or Mch5 containing nucleotide sequences, antibody or ligandmediated detection with Mch4 or Mch5 binding agents or by enzymecatalysis of detectable Mch4 or Mch5 substrates. Such methods areroutine to those skilled in the art. Detection can be performed ex vivo,for example, by removing a cell or tissue sample from an individualexhibiting or suspected of exhibiting a cell death mediated disease.Correlation of increased Mch4 or Mch5 expression or activity isindicative of diseases characterized by enhanced programmed cell deathwhereas correlation of decreased Mch4 or Mch5 expression or activity isindicative of diseases characterized by the inhibition of programmedcell death.

The above Mch4 or Mch5 polypeptides can also be formulated intopharmaceutical compositions known within the art for the treatment ofcell death mediated diseases characterized by increased cell survivaland proliferation. Functional fragments and peptides such as thecatalytic domain of Mch4 or Mch5 can similarly be formulated for thetreatment of such diseases associated with increased cell survival andproliferation. Administration of Mch4 or Mch5 polypeptides andfunctional fragments thereof will induce apoptosis in treated cells andeliminate those cells characterized by increased cell survival orproliferation. Administration of non-Mch4 or Mch5 polypeptides that donot directly act on Mch4 or Mch5 substrates but induce the activation ofthe Mch4 or Mch5 protease can similarly be used for the treatment ofdiseases characterized by increased cell survival and proliferation.

To be effective, the Mch4 or Mch5 polypeptides must be introduced intothe cells characterized by increased cell survival. Introduction can beaccomplished by a variety of means known within the art including, forexample, lipid vesicles and receptor mediated endocytosis. Targeting tothe appropriate cell type can similarly be accomplished throughconjugation to specific receptor ligands, specific target cellantibodies and the like.

The Mch4 or Mch5 polypeptides are administered by conventional methods,in dosages which are sufficient to induce apoptosis in the cellscharacterized by increased cell survival or proliferation. Such dosagesare known or can be easily determined by those skilled in the art.Administration can be accomplished by, for example, intravenous,interperitonal or subcutaneous injection. Administration can beperformed in a variety of different regimes which include single highdose administration or repeated small dose administration or acombination of both. The dosing will depend on the cell type,progression of the disease and overall health of the individual and willbe known or can be determined by those skilled in the art.

In contrast to the induction of Mch4 or Mch5 mediated apoptosis for thetreatment of pathological conditions characterized by increased cellsurvival or proliferation, inhibitors of Mch4 or Mch5 can be used totreat diseases characterized by increased programmed cell death. Suchinhibitors can be, for example, anti-Mch4 or anti-Mch5 antibodies,proteins, or small peptidyl protease inhibitors, or small non-peptide,organic molecule inhibitors which are formulated in a medium whichallows introduction into the desired cell type. Alternatively, suchinhibitors can be attached to targeting ligands for introduction by cellmediated endocytosis and other receptor mediated events. Specificexamples of Mch4 or Mch5 peptidyl inhibitors are described in Table I ofExample III and includes suicide inhibitors and substrate analogues suchas the tetrapeptide DEVD aldehyde and the cowpox virus protein Crm A,for example.

Other inhibitors of Mch4 or Mch5 include, for example, small moleculesand organic compounds which bind and inactivate Mch4 or Mch5 by acompetitive or non-competitive type mechanism. Molecules or compoundswhich indirectly inhibit the Mch4 or Mch5 pathway can also be used asinhibitors of Mch4. Mch4 or Mch5 inhibitors can be identified byscreening for molecules which demonstrate specific or beneficial Mch4 orMch5 inhibitory activity. Such methods are described further below andcan be practiced by those skilled in the art given the Mch4 or Mch5nucleotide and amino acid sequences described herein.

Dominant/negative inhibitors of Mch4 or Mch5 can also be used to treator reduce the severity of diseases characterized by increased programmedcell death. In this regard, Mch4 or Mch5 large subunits which lack theactive site QACQG can be used to bind the small subunits of Mch4 or Mch5and prevent active protease complexes from forming. Such a mechanism ofdominant negative inhibition of Mch4 is similar to the dominant negativeinhibition of Ich-1_(L) by Ich-1_(S). Subunits from other ASCPs cansimilarly be used as dominant/negative inhibitors of Mch4 or Mch5activity and therefore treat diseases mediated by programmed cell death.Such subunits should be selected so that they bind either the p17 or p12Mch4 or Mch5 polypeptides and prevent their assembly into activetetrameric protease complexes. Moreover, Mch4 or Mch5 subunits whichhave been modified so as to be catalytically inactive can also be usedas dominant negative inhibitors of Mch4. Such modifications include, forexample, mutation of the active site cysteine residue to include but notlimited to Alanine or glycine.

Mch4 or Mch5 substrate antagonists can similarly be used to treat orreduce the severity of diseases mediated by increased programmed celldeath. Such substrate antagonists can bind to and inhibit cleavage byMch4. Inhibition of substrate cleavage prevents commitment progressionof programmed cell death. Substrate antagonists include, for example,ligands and small molecule compounds.

Treatment or reduction of the severity of cell death mediated diseasescan also be accomplished by introducing expressible nucleic acidsencoding Mch4 or Mch5 polypeptides or functional fragments thereof intocells characterized by such diseases. For example, elevated synthesisrates of Mch4 or Mch5 can be achieved by, for example, using recombinantexpression vectors and gene transfer technology. Similarly, treatment orreduction of the severity of cell death mediated diseases can also beaccomplished by introducing and expressing antisense Mch4 or Mch5nucleic acids so as to inhibit the synthesis rates of Mch4 or Mch5. Suchmethods are well known within the art and will be described below withreference to recombinant viral vectors. Other vectors compatible withthe appropriate targeted cell can accomplish the same goal and thereforecan be substituted in the methods described herein in place ofrecombinant viral vectors.

Recombinant viral vectors are useful for in vivo expression of a desirednucleic acid because they offer advantages such as lateral infection andtargeting specificity. Lateral infection is inherent in the lifecycle ofretroviruses and is the process by which a single infected cell producesmany progeny virions that bud off and infect neighboring cells. Theresult is a large area becomes rapidly infected, most of which were notinitially infected by the original viral particles. This is in contrastto vertical-type of infection in which the infectious agent spreads onlythrough daughter progeny. Viral vectors can also be produced that areunable to spread laterally. This characteristic can be useful if thedesired purpose is to introduce a specified gene into only a localizednumber of targeted cells.

Typically, viruses infect and propagate in specific cell types.Therefore, the targeting specificity of viral vectors utilizes thisnatural specificity to in turn specifically introduce a desired geneinto predetermined cell types. The vector to be used in the methods ofthe invention will depend on desired cell type to be targeted. Forexample, if neurodegenerative diseases are to be treated by decreasingthe Mch4 or Mch5 activity of affected neuronal cells then a vectorspecific for cells of the neuronal cell lineage should be used.Likewise, if diseases or pathological conditions of the hematopoieticsystem are to be treated, than a viral vector that is specific for bloodcells and their precursors, preferably for the specific type ofhematopoietic cell, should be used. Moreover, such vectors canadditionally be modified with specific receptors or ligands and the liketo modify or alter target specificity through receptor mediated events.These modification procedures can be performed by, for example,recombinant DNA techniques or synthetic chemistry procedures. Thespecific type of vector will depend upon the intended application. Theactual vectors are also known and readily available within the art orcan be constructed by one skilled in the art using well knownmethodology.

Viral vectors encoding Mch4 or Mch5 nucleic acids or inhibitors of Mch4or Mch5 such as antisense nucleic acids can be administered in severalways to obtain expression of such sequences and therefore eitherincrease or decrease the activity of Mch4 or Mch5 in the cells affectedby the disease or pathological condition. If viral vectors are used, forexample, the procedure can take advantage of their target specificityand consequently, do not have to be administered locally at the diseasedsite. However, local administration can provide a quicker and moreeffective treatment. Administration can also be performed by, forexample, intravenous or subcutaneous injection into the subject.Injection of the viral vectors into the spinal fluid can also be used asa mode of administration, especially in the case of neurodegenerativediseases. Following injection, the viral vectors will circulate untilthey recognize host cells with the appropriate target specificity forinfection.

As described above, one mode of administration of Mch4 or Mch5 encodingvectors can be by direct inoculation locally at the site of the diseaseor pathological condition. Local administration is advantageous becausethere is no dilution effect and therefore a smaller dose is required toachieve Mch4 or Mch5 expression in a majority of the targeted cells.Additionally, local inoculation can alleviate the targeting requirementrequired with other forms of administration since a vector can be usedthat infects all cells in the inoculated area. If expression is desiredin only a specific subset of cells within the inoculated area thenpromoter and expression elements that are specific for the desiredsubset can be used to accomplish this goal. Such non-targeting vectorscan be, for example, viral vectors, viral genomes, plasmids, phagemidsand the like. Transfection vehicles such as liposomes can be used tointroduce the non-viral vectors described above into recipient cellswithin the inoculated area. Such transfection vehicles are known by oneskilled within the art. Alternatively, however, non-targeting vectorscan be administered directly into a tissue of any individual. Suchmethods are known within the art and are described by, for example,Wolff et al. (Science 247:1465-1468 (1990)).

Additional features can be added to the vectors to ensure safety and/orenhance therapeutic efficacy. Such features include, for example,markers that can be used to negatively select against cells infectedwith the recombinant virus. An example of such a negative selectionmarker is the TK gene described above that confers sensitivity to theantibiotic gancyclovir. Negative selection is therefore a means by whichinfection can be controlled because it provides inducible suicidethrough the addition of antibiotic. Such protection ensures that if, forexample, mutations arise that produce mutant forms of Mch4 or Mch5,dysfunction of apoptosis will not occur.

As described previously, the Mch4 or Mch5 encoding nucleic acids andMch4 or Mch5 polypeptides of the invention can be used to screen forcompounds which inhibit or enhance the expression of Mch4 or Mch5protease activity. Such screening methods are known to those skilled inthe art and can be performed by either in vitro or in vivo procedures.For example, described in Example II is a specific in vitro assay forMch4 or Mch5 activity. This assay employs Mch4 or Mch5 polypeptideexpressed in an active, processed form recombinantly in E. coli, whoseprotease activity is measured by incubation with a fluorescent substrate(DEVD-AMC). Also described therein are peptide and polypeptideinhibitors of Mch4. This assay can be used to screen synthetic ornaturally occurring compound libraries, including macromolecules, foragents which either inhibit or enhance Mch4 or Mch5 activity. The Mch4or Mch5 polypeptides to be used in the assay can be obtained by, forexample, in vitro translation, recombinant expression or biochemicalprocedures. Methods other than that described in Example II can also beused to screen and identify compounds which inhibit Mch4. A specificexample is phage display peptide libraries where greater than 10⁸peptide sequences can be screened in a single round of panning. Suchmethods as well as others are known within the art and can be utilizedto identify compounds which inhibit or enhance Mch4 or Mch5 activity.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Cloning And Characterization of Mch4

This Example shows the cloning, sequence analysis and tissuedistribution of Mch4 and Mch5. The results described herein indicatethat Mch4 and Mch5 are novel members of the cell death family ofaspartate-specific cysteine proteases.

To identify potentially novel members of the ICE family of cysteineproteases, an approach combining information from the GenBank databaseof human expressed sequence tags (ESTs) and PCR was employed. Initially,Ced-3/ICE-like apoptotic cysteine proteases from Jurkat T-lymphocyteswere enriched by amplification of a human Jurkat cDNA library usingdegenerate PCR primers encoding the conserved GSWFI/GSWYI pentapeptides(Fernandes-Alnermi et al., Cancer Res. 55:2737-2742 (1995a)). This aminoacid sequence has been found to be conserved among ICE family members.Briefly, a 10 μl aliquot of human Jurkat λ Uni-Zap™ XR cDNA librarycontaining approximately 10⁸ pfu was denatured at 99° C. for 5 min. andused as a substrate for PCR amplification with a degenerate primerencoding the pentapeptide GSWFI/GSWYI and a T3 vector-specific primer(Stratagene).

The enriched library was then amplified with a primer derived from anEST sequence identified in a homology search of the GenBank databaseusing a query nucleotide sequence corresponding to the Mch2 and CPP32coding sequence. The secondary amplification was performed starting witha 10 μl aliquot of the above amplified sequences combined with a primerderived from the GenBank sequence T96912 (primer T96-pr1:TCAGCCTCGGCAGGAATAC SEQ ID NO:5) and a second vector specific primer(SK-Zap: CAGGAATTCGGCACGAG, SEQ ID NO:6). The secondary amplificationproducts were cloned into a Sma I cut pBluescript II KS⁺ vector. Allclones were screened by PCR using a degenerate oligonucleotidecorresponding to the conserved active site amino acid sequence QACRG andthe SK-Zap primer. Clones that were positive for the presence of theQACRG coding sequence were then subjected to DNA sequencing using T3 andT7 sequencing primers (Stratagene). This amplification and screenresulted in the identification of a Ced-3/ICE-like partial cDNA withhigh homology to CPP32 and Ced-3.

Partial cDNA identified from the QACRG screening was then excised fromthe vector, radiolabeled and used to screen the original Jurkat 80Uni-Zap™ XR cDNA library for full length cDNA clones. Positive 80 cloneswere purified, rescued into the pBluescript II SK⁻ plasmid vector andsequenced.

The above screening identified a 3.6 kb cDNA clone from the human JurkatT-lymphocyte cDNA library. This cDNA contains an open reading frame of1038 bp that encodes a 346-amino acid protein, named Mch4 (SEQ ID NOS:1and 2, respectively). As shown in FIGS. 1 and 3A, Mch 4 is a polypeptideof 346 amino acid residues with a predicted molecular mass of 39 kDa.Although discussed more fully below in regard to the tissuedistribution, the Mch4 polypeptide is encoded by an approximately 4.0 kbmRNA. This size, together with the presence of an in-frame stop codon184 bp upstream from the initiator methionine indicates that the clonedMch4 cDNA (SEQ ID NO:1) contains the full length coding region.

Following identification of and cloning of Mch4, a subsequent search ofthe GenBank database resulted in the identification of a second novelEST sequence (N42544) with extensive homology to Mch4. Briefly, ahomology search of the GenBank database of human expressed sequence tags(ESTs) for sequences similar to Mch4 revealed a 449 bp EST sequence(N42544) with a 64% identity to Mch4. Using PCR primers derived fromthat EST sequence (Mch5-pr1, GACAGAGCGAGATTCTGT; Mch5-pr2,GCACCATCAATCAGAAGG (SEQ ID NOS:7 and 8, respectfully)) and the vectorspecific primers T3 and SK-Zap, the full length cDNA corresponding tothis gene was amplified by PCR from the Jurkat cDNA library and clonedin KS-vector. To perform this amplification, the Mch5-pr1 and the T3vector primers were used for the primary PCR amplification step toamplify 5' sequences of Mch5 while the Mch5-pr2 and the SK-Zap vectorspecific primer was used for the secondary amplification step. The fulllength cDNA was sequenced and its gene product was named Mch5 (SEQ IDNO:3).

The Mch5 cDNA encodes a 389 amino acid protein (SEQ ID NO:4) with thehighest degree of homology to Mch4 compared to other family members. Acomparison of the amino acid sequence identities between Mch4 and Mch5is shown in FIG. 3A while a multiple amino acid sequence alignment ofall known ASCPs is shown in FIG. 3B (SEQ ID NOS:12-61). Although thesequence comparisons of Mch4 and Mch5 are discussed further below. Theoverall sequence identity between Mch4 and Mch5 is 46%. These resultsindicate that Mch4 and Mch5 are in fact distinct ASCPs and not variantsof a single gene product.

The identification and sequence analysis of the novel apoptoticproteases described herein has now revealed that both Mch4 and Mch5belong to the Ced-3-like subfamily of ASCPs. Briefly, previouslyidentified ASCPs can be divided phylogentically into three subfamilies.The Ced-3-like ASCP subfamily includes Ced-3, CPP32, Mch2, and Mch3 (SEQID NOS:9-12, 37-41, 32-36 and 27-31, respectively). The ICE-like ASCPsubfamily includes ICE, TX(ICH2, ICErel-II, Mih1) and ICErelIII (SEQ IDNOS:42-46, 47-51 and 52-56, respectively). The NEDD-like subfamilyinclude ICH-1 and its mouse counterpart NEDD2 (SEQ ID NO:16). Sequencealignment of Mch4 (SEQ ID NOS:12-16) and Mch5 (SEQ ID NOS:17-21) withthese known ASCPs is shown in FIG. 3B and reveals both of these newASCPs belong to the Ced-3-like subfamily of ASCPs.

Shown in FIG. 3B is a multiple amino acid sequence alignment ofrelatively conserved regions within the ASCPs. These regions include,for example, (1) the active site pentapeptide QACRG, (2) the substratebinding residues P1-P4 and (3) the putative processing sites between thesmall and large subunits. Relevant sequence comparisons for each ofthese regions as well as other worthy distinctions is discussed morefully below.

For example, in the region that does not contain the propeptide domain,Mch4 and Mch5 are equally related to Ced-3 (SEQ ID NOS:37-41) exhibitingan overall amino acid identity of 32% and sequence similarity of 54%.Comparing with the other human Ced-3-like subfamily members, Mch4 ismore related to Mch2 and Mch3 (SEQ ID NOS: 27-31 and 22-26,respectively) with a 38-40% sequence identity and a 56-58% similaritythan it is to CPP32 (SEQ ID NOS:32-36). The latter comparison revealinga 35% amino acid identity and a 57% amino acid sequence similarity. Onthe other hand, Mch5 is equally related to CPP32, Mch2 and Mch3 with a39-40% amino acid sequence identity and a 60-62% sequence similarity.

Comparison of Mch4 and Mch5 reveals a significant degree of homologywith an overall sequence identity of 52% and similarity of 67% at theprimary amino acid level excluding the propeptide domain. As shown inFIG. 3B, the homology between the two proteins is highest within thesmall subunit region. A similar relationship was observed with otherfamily members such as CPP32/Mch3 and ICE/TX. These sequencesimilarities indicate that Mch4 and Mch5 similarly likely interact witheach other as do their related family members CPP32 and Mch3(Fernandes-Alnemri et al., Cancer Res. 55:6045-6052 (1995b)). Forexample, Mch4 and Mch5 likely heterodimerize with each other to formfunctional protease heterocomplexes as do CPP32 and Mch3.

Sequence alignment also revealed that, although distinct, Mch4 and Mch5are structurally similar to other known ASCPs. The active enzymes ofMch4 and Mch5 are made of two subunits, derived from precursorproenzymes (proMch4 and proMch5) by cleavage at highly conserved Aspresidues (Asp239 in Mch4 and Asp284 in Mch5) located between the twosubunits (denoted as D/X in FIG. 3B). Consistent with other ASCPS,ProMch4 and proMch5 are likely processed further to remove thepropeptide domains. Several aspartate cleavage sites are present in theprodomain region of both Mch4 and Mch5 (FIG. 3A).

Regardless of the above similarities, one major difference between Mch4and Mch5 and other family members is that their active site pentapeptidecontains an Arg to Gln non-conservative substitution. The substitutionchanges the previously conserved peptapeptide sequence from QACRG toQACQG. Such a substitution could have major effects on enzyme andsubstrate specificities. The presence of QACQG instead of QACRG in thesetwo enzymes, suggests that other unknown family members with a similarsubstitution may exist. This result further increases the complexity ofthe ASCP family.

In regard to specific amino acid residues that have been implicated toplay functional roles, the crystal structure of ICE has indicated thatthe amino acid residues His237, Gly238 and Cys285 are involved incatalysis, while Arg179, Gln283, Arg341 and Ser347 are involved inbinding the carboxylate side chain of the substrate P1 aspartate. Withthe exception of Ser347 in Mch5, all of these other residues areabsolutely conserved in all family members. Nevertheless, the Ser to Thrsubstitution in Mch5 corresponding to Ser347, is a conservativesubstitution and it is the only one among all the family members (FIG.3B). Another Ser to Thr conservative substitution can also be seen inMch4 in the region corresponding to Ser236. This residue is one thatparticipates in binding the substrate P2-P4 residues. However, othersresidues that might participate in binding the substrate P2-P4 residuesare not widely conserved. This result indicates that these otherresidues likely determine substrate specificity.

EXAMPLE II Tissue Distribution And Chromosomal Localization of Mch4

This Example shows the expression pattern of Mch4 as measured by RNAblot analysis and the genetic locus of the Mch4 gene.

The tissue distribution of Mch4 was analyzed by RNA blot analysis ofpoly A⁺ RNA isolated from different human tissues. Briefly, tissuedistribution analysis of Mch4 MRNA was performed on RNA blots preparedby Clontech (San Diego, California) containing 2 μg/lane of poly A⁺ RNAfrom each tissue of origin. A radioactive Mch4 riboprobe was preparedusing Mch4 cDNA as a template for T7 RNA polymerase in the presence ofα³² P! ATP. The blots were hybridized, washed and then visualized byautoradiography.

The results of the RNA blots revealed that a major 3.7 Kb Mch4 messagewas detectable in most tissues examined. The lowest expression of Mch4mRNA was seen in whole brain, kidney, prostate, testis and colon. Thesize of the Mch4 mRNA is consistent with the length of the cloned Mch4cDNA (3.6 kb). Other higher molecular weight mRNA species can also beseen in some tissues such as skeletal muscle, for example, and couldrepresent unprocessed Mch4 mRNA or an mRNA of a related family member.

To determine the chromosomal localization of the Mch4 gene, a panel ofrodent-human somatic cell hybrids was screened by PCR with Mch4 specificprimers. Briefly, A panel of DNAs from rodent-human somatic cell hybridswas screened by PCR with the previously described Mch4 specific primerst96-pr1 (SEQ ID NO:5) and a second Mch4 specific primer termed t96-pr5(CGGGAGATCATGTCTCAC, SEQ ID NO:17). These primers were also used toscreen by PCR the CEPH A and B YAC libraries.

The results of these searches identified two YAC clones (756A9 and800G4) which were positive for Mch4. A computer search through theWhitehead Institute and CEPH databases showed that both YACS were partof the WI contigs WC-630 and Wc2.16 and of the CEPH contig at position2.08 of chromosome 2. Other YACS (741D10, 762C12, 809H8, and 828E8)reported by the databases to overlap with 756A9 and/or 800G4 were testedby PCR for the presence of MCH4 gene sequences. Clones 762C12 and 828E8were found to be positive for MCH4. This analysis resulted in theassignment of Mch4 to chromosome 2p12-qter. To confirm these mappingresults and to obtain a definite physical localization for the MCH4gene, the non-chimeric YAC 828E8 was used in FISH analysis to probenormal human lymphocyte metaphases. The Mch4 chromosomal localizationwas narrowed to chromosome 2q33-34 using his latter analysis. Thisplaces the Mch4 gene within a 4 cM region flanked by the centromericmarker D2S374 and the telomeric marker D2S346 (Chumakov et al., Nature377(supp.):175-183 (1995)).

EXAMPLE III Kinetic Parameters of Mch4

This Example characterizes the protease activity and substratespecificity of the ASCP Mch4.

The kinetic properties of bacterially expressed recombinant Mch4 weredetermined using the tetrapeptide substrates DEVD-AMC and YVAD-AMC in acontinuous fluorometric assay (Table I). The DEVD-AMC and the YVAD-AMCrepresent the cleavage sites for the poly(ADP-ribose)polymerase (PARP)and IL-1β P1-P4 substrate tetrapeptides, respectively (Nicholson et al.,Nature 376:37-43 (1995)). Briefly, Mch4 cDNA lacking most of thepropeptide coding sequence (amino acids 61-346) was subcloned in-frameinto the Bam HI/XhoI sites of the bacterial expression vector pGEX-5X-3(Pharmacia Biotech Inc.). This vector produces Mch4 as a fusion proteinwith glutathione S-transferase (GST) and was used essentially asdescribed in Fernades-Alnemri et al., supra (1995a). The GST-Mch4expression vector was constructed and transformed into DH5α bacteriausing routine molecular biology methods known to those skilled in theart. After induction with IPTG, bacterial extracts were prepared from E.coli expressing the recombinant fusion proteins. The extracts wereadsorbed to glutathione-Sepharose resin, washed several times and thenanalyzed by SDS-PAGE. The Mch4 preparation contained a protein thatmigrated as a doublet of approximately 50 kDa (GST-large subunit fusion)and 12 kDa (small subunit).

The purified Mch4 GST-fusion protein was then used for further enzymaticanalyses. The activity of Mch4 was measured using bacterial lysatesprepared with ICE buffer (25 mM HEPES, 1 mM EDTA, 5 mM DTT, 0.1% CHAPS,10% sucrose, pH 7.5) at room temperature (24-25° C). The K_(i) 's weredetermined from the hydrolysis rate of 50 μM DEVD-AMC following a 30 minpreincubation of the enzyme with inhibitors DEVD-CHO and recombinantCrmA protein. Prior to incubation with enzyme, purified CrmA wasactivated by incubation with 5 mM DTT for 10 min at 37° C.

                  TABLE I                                                         ______________________________________                                        Kinetic Parameters of Mch4                                                    Parameter             Value                                                   ______________________________________                                        K.sub.m (DEVD-AMC)    130    μM                                            K.sub.m (YVAD-AMC)    150    μM                                            K.sub.i (DEVD-CHO)    14     nM                                               K.sub.i (CrmA)        0.75   μM                                            ______________________________________                                    

As shown above in Table I, the K_(m) values of Mch4 for the two peptidesubstrates DEVD-AMC and YVAD-AMC are similar. These values contrast withthose for CPP32, where the K_(M) for the YVAD-AMC substrate is >35-foldhigher than the K_(M) for the DEVD-AMC substrate (Fernandes-Alnemri etal. supra (1995b)). These kinetic references are further illustrated bythe ratio of Vmax/Km for the DEVD-AMC substrate. Specifically, CPP32possesses a >500-fold higher specificity for this substrate compared toMch4 (V_(max) K_(mCPP32) =9200 and V_(max) /K_(mMch4) =18). However,similar to CPP32 and Mch3α, Mch4 is potently inhibited by the DEVD-CHOpeptide (K_(iMch) 4=14 nM) and weakly inhibited by Crm A (K_(iMch4)=0.75 μM) (Fernandes-Alnemri et al., supra (1995b)). Since DEVD-CHO alsoblocks cell death, this result further indicates that Mch4 is an ASCPwhich plays a role in the cell death pathway.

EXAMPLE IV Granzyme B Activates Multiple Members of the Mammalian CED-3Subfamily

This Example shows that the cytotoxic T cell protease essential forinduction of apoptosis in target cells directly activates ASCP membersof the Ced-3 subfamily by cleavage into the large and small proteasesubunits.

Granzyme B has been shown to cleave CPP32 to generate an ˜20 kDacleavage product presumed to be the large subunit of CPP32 (Darmon etal., Nature 377:446-448 (1995)). This cleavage event has attracted theidea that the granzyme B cleavage occurs at the processing sequenceIETD-S between the two subunits of CPP32 (FIG. 3B). Sequence comparisonof the Mch4 and Mch5 ASCPs described herein has revealed that thepotential processing sequences between the two subunits of Mch3 and Mch4are very similar to that of CPP32 (FIG. 3B). These two sequences containidentical P1 residues (CPP32-D175, Mch3-D198, Mch4-D239) and P4 residues(CPP32-I172, Mch3-I195, Mch4-I236) in all three proenzymes and aconserved P3 residue (CPP32-E173, Mch3-Q197, Mch4-E237), suggesting thatif the processing site in CPP32 is in fact cleaved by granzyme B, thenthese other subfamily members may similarly be substrates for cleavageas well.

To determine whether granzyme B can cleave these proenzymes at theproposed processing sites, mutant proenzymes with a P1 substitutionmutation converting D to A in CPP32 and Mch3 or a D to G in Mch4 weregenerated. Briefly, potential aspartate processing sites between the twosubunits of these ASCPs were mutated to alanine (CPP32 and Mch3) orglycine (Mch4) by site directed mutagenesis using overlapping PCRmutagenic oligonucleotides. Two internal mutagenic overlappingoligonucleotide primers encoding the D/A or the D/G mutation and twoexternal oligonucleotides encoding the first six N-terminal amino acidsand last six C-terminal amino acids, respectively, were used in a PCRreaction with CPP32, Mch3 and Mch4 cDNAs. Asp9 of proCPP32 was mutatedto Ala by PCR using a 5' mutagenic oligonucleotide encoding the D to Amutation and a 3'-primer derived from the 3'-noncoding sequence of CPP32cDNA. The resulting PCR products were subcloned in pBluscript II KS⁺vector under the T7 promoter and their sequence was verified by DNAsequencing.

Wild type and mutated cDNAs were in vitro transcribed and translated inthe presence of ³⁵ S-methionine using Promega coupledtranscription/translation TNT kit according to the manufacturerrecommendations. Two microliters of the translation reactions wereincubated with purified enzymes (100-200 ng) or bacterial lysatesexpressing recombinant ASCPs in ICE-buffer, in a final volume of 10 μl.The reaction was incubated at 37° C. for 1-2 hours and then analyzed bySDS-PAGE and autoradiography.

Following in vitro translation, the parental and mutant proenzymes wereincubated with granzyme B and then analyzed by SDS-PAGE andautoradiography. As shown in FIG. 4A, in vitro translation of wild type(lane 1) or AsP175-mutated (lanes 2 and 3) CPP32 proenzymes generatedidentical pattern of translation products. The 15 major translationproducts started with Met27 and Met39, respectively. Incubation of thesetranslation products with granzyme B resulted in cleavage of the wildtype proCPP32 at Asp175 (lane 5) to generate the two subunits of activeCPP32. The small C-terminal subunit migrates as a single ˜12 kDa bandand the large N-terminal subunit migrates as three bands (˜21, ˜19 and˜17 kDa).

In regard to the identities of the bands comprising the large subunit,the faint ˜21 kDa band is most likely a cleavage product of the fulllength proCPP32. However, the high intensity of the ˜19 kDa bandsuggests that it is produced from the ˜21 kDa band by further processingat the propeptide domain. This indication is supported by theobservation that incubation of proCPP32 with granzyme B in the presenceof the CPP32 peptide inhibitor DEVD-CHO, generated a major ˜21 kDa bandwhich was not further processed to the ˜19 kDa band (FIG. 4B, lane 8).Further processing of the ˜21 kDa band to the ˜19 kDa band was onlyobserved in the absence of the peptide inhibitor DEVD-CHO (FIG. 4A, lane5 and FIG. 4B, lane 9). This result indicates that the additionalprocessing of the propeptide domain seen with the wild type proCPP32 isdue to the autocatalytic activity of the granzyme B-activated CPP32. Nocleavage was observed with the buffer control or the AsP175-mutatedCPP32 (FIG. 4A, lanes 1 and 3, respectively).

In addition there was no cleavage at the propeptide domain of theAsP175-mutated CPP32 (FIG. 4A, lane 2 and FIG. 4B, lane 3), indicatingfurther support to our earlier conclusion that cleavage of thepropeptide is an autocatalytic activity of activated CPP32. Furthermore,the autocatalytic processing within the propeptide domain occurs at Asp9and not at Asp28. Mutation of Asp9 to Ala inhibited the processing ofthe ˜21 kDa band to the ˜19 kDa band in a similar fashion as observedwith the DEVD-CHO inhibitor (FIG. 4B, lanes 5 and 6). These dataindicate that CPP32 is autocatalytically processed at Asp9 afteractivation to generate a p19 (large subunit) and p12 (small subunit).

Earlier observation that purified human CPP32 was processed at Asp28,could be due to the fact that CPP32 was purified from THP-1 monocytecytosol after incubation at 37° C. for several hours (Nicholson et al.,supra (1995)). THP-1 cytosol contains high concentration of ICE andpossibly other ICE homologs that might be responsible for the additionalprocessing at Asp28. The ˜17 kDa band is a cleavage product of one ofthe smaller internally translated products, most likely the 29 kDa band.

Similarly, in vitro translation of wild type or Asp198-mutated proMch3(FIG. 5A, lanes 1 and 2, respectively) generated two major products.These two translation products are a 35-36 kDa product corresponding tothe full length proMch3 and a 30 kDa internal translation productionmost likely starting with Met45. Other internal translation productssmaller than 30 kDa can also be seen.

Like proCPP32, incubation of proMch3 with granzyme B generated the twosubunits of the active Mch3 enzyme (FIG. 5A, lane 6). These subunits canbe seen as a ˜12 kDa band corresponding to the small C-terminal subunitand two ˜20 and ˜18 kDa bands corresponding to the large N-terminalsubunit. The ˜20 kDa band is a product of processing at Asp198 betweenthe two subunits, and at Asp23 in the propeptide domain. The ˜18 kDaband is a cleavage product of the smaller 30 kDa internally translatedproduct. No cleavage products corresponding to the small or largesubunits were observed with the buffer control or the Asp198-mutatedproMch3 (FIG. 5A, lanes 1, 2 and 4, respectively).

Unlike CPP32, there was a 33 kDa cleavage product in the Asp198-mutatedproMch3 (lane 4). This product is a result of granzyme B cleavage in thepropeptide domain of proMch3, indicating that granzyme B can processproMch3 to active Mch3 without the requirement of an additional activityto remove the propeptide domain. Nevertheless, we have shown recentlythat CPP32 can also cleave the propeptide domain of proMch3 veryefficiently (Fernandes-Alnemri et al., supra (1995b)). Consequently,activation of CPP32 in vivo by granzyme B would result in furtherprocessing of both CPP32 and its closely related homolog Mch3.

In vitro translation of wild type or Asp239-mutated proMch4 (FIG. 5B,lanes 4 and 5, respectfully) generated two major products. These twotranslation products are observed as a 39 kDa product corresponding tothe full length proMch4 and a 27 kDa internal translation product. Theinternally translated product starts with Met102. This was confirmed bydeletion of the cDNA sequence encoding the first 101 amino acids andallowing the translation to proceed from Met102. This deletion produceda truncated Mch4 protein that was similar in size to the internallytranslated ˜27 kDa Mch4 protein (FIG. 5B, lane 1).

Granzyme B cleaved the truncated Mch4 to generate 15-16 kDa and 12 kDabands (FIG. 5B, lane 3). On the other hand, Granzyme B cleaved the fulllength Mch4 to generate ˜27 kDa band (large subunit) and 12 kDa band(small subunit) (lane 8). However, because of the presence of theinternally translated 27 kDa protein together with the full length Mch4,a 15-16 kDa band was also produced after incubation with granzyme B(lane 8). Like CPP32 and Mch3, the Asp239-mutated Mch4 was not cleavedby granzyme B (lane 6), and there was no cleavage in the buffer control(lanes 1 and 4).

These data show that granzyme B not only activates pro-CPP32, but alsothe related ASCPs Mch3 and Mch4 by cleavage at the IETD-S, IQAD-A andIEAD-A putative processing sequences, respectively. Cleavage at thesesites generates the two subunits that form the active enzyme complex ofthese proteases.

EXAMPLE V Mch4 is Upstream of CPP32 and Mch3 in the ASCP Cascade

This Example shows that Mch4 is capable of activating both proCPP32 andproMch3 while remaining resistant to cleavage from its proenzyme statewhen incubated in the presence of activated forms of either CPP32 orMch3.

Evidence suggests that ASCPs are involved in a cascade of activationevents which leads to the final cell death signal. To determine whethersuch a cascade exists within the Ced-3 subfamily of ASCPs occurs, theactivation of CPP32 and its closely related homolog Mch3 by anothersubfamily member such as Mch4 was assessed. Activation was determined byincubating purified recombinant Mch4 with proCPP32 and proMch3.

Analysis of the cleavage products showed that Mch4 processed proCPP32and proMch3 and generated cleavage products identical to those producedby granzyme B (FIG. 4A, lane 4 and FIG. 5A, lane 5). Mch4 was unable toprocess the Asp to Ala mutated proCPP32 and proMch3 (FIG. 4A, lane 3 andFIG. 5A, lane 3). However, like granzyme B, Mch4 was able to cleave thepropeptide of Mch3 to generate a 33 kDa band (FIG. 5A, lane 3). AlthoughMch4 was able to cleave proMch4, its activity towards its proenzyme wassignificantly lower than that towards proCPP32 and proMch3 (FIG. 5B,lane 7). In addition there was no significant cleavage of proMch4 whenincubated with recombinant CPP32 or Mch3 enzymes (FIG. 6). The activityof several other ASCPs such as ICE, TX and Mch2, were also tested butnone of these enzymes were able to efficiently process Mch4.

These data indicate that Mch4 is upstream of CPP32 and Mch3 in theapoptotic protease cascade.

The above results indicating that Mch4, Mch3 and CPP32 play a role in aprotease cascade are further supported by the unique features exhibitedby these and related ASCPs. Specifically, ASCPs have two unique featuresthat distinguish them from other proteases. First, they all cleave theirsubstrates after Asp residues, and their activation requires cleavageafter Asp residues located in highly conserved processing sites betweentheir large and small subunits. The ability to cleave after Asp residuesis only shared with granzyme B, a serine protease that does not,however, require cleavage after Asp residues for its activation.

The above features indicate that ASCPs interact with and activate eachother in a protease cascade fashion as well as acting as substrates forgranzyme B. In addition, because multiple ASCP family members coexist inone cell type the ability of one family member to activate several otherfamily members and vice versa results in multiple protease cascades andthe generation of multiple apoptotic pathways. Evidence for theexistence of multiple apoptotic pathways is corroborated from studieswith mice deficient in ICE or Bcl2. For example, thymocytes from ICEdeficient mice remain sensitive to glucocorticoid- and ionizingradiation-induced apoptosis, but become resistant to antiFas-inducedapoptosis (Kuida et al., Science 267:2000-2003 (1995)). On the otherhand, T-cells from bcl2 deficient mice become more sensitive toglucoccorticoid- and ionizing radiation-induced apoptosis, but lesssensitive to antiCD3-induced apoptosis.

As shown in FIG. 7, the above results indicate the existence of multipleprotease cascades that can be activated by different apoptotic stimuli.For example, one of these cascades involves Mch4 acting upstream ofCPP32, Mch2 and Mch3. Once Mch4 is activated by certain apoptoticstimuli, it can process and activate the proenzymes of Mch3 and CPP32 asshown above. These two ASCPs are likely responsible for PARP cleavage inapoptosis. Active CPP32 can in turn activate proMch2, the only ASCP thatcan cleave lamin. Because CPP32, Mch3 and Mch4 are poorly inhibited byCrm A (see Table I), the above cascade would not be affected inICE-knockout mice, or be inhibited by the ICE inhibitor Crm A.Therefore, it is likely that glucocortiocoid- and radiation-inducedapoptosis occur through this cascade.

In an alternative ICE-like cascade, activation of ICE by an apoptoticstimulus results in the activation of CPP32, Mch2 and Mch3 (FIG. 7).This activation results because ICE can activate proCPP32 (Tewari etal., Cell 81:801-809 (1995)). In yet another distinct apoptotic proteasecascade an exogenous protease is used to activate multiple endogenousASCPs. This is the granzyme B-cascade which is used by cytotoxicT-lymphocytes to kill their target cells (see FIG. 7). With theunderstanding of these multiple cascades and their regulatory activationevents, it is now possible to target these pathways either alone or incombination for the therapeutic treatment of human diseases.

EXAMPLE VI Mch4 Exhibits Cell Death Activity

This Example shows the expression of Mch4 and induction of apoptosis incultured cells.

To determine if Mch4 exhibits cell death activity, the induction ofearly apoptosis in Sf9 baculovirus cells was assessed. Briefly, Sf9cells were infected with recombinant baculoviruses encoding full lengthMch4 or full length CPP32 as a standard (Fernandes-Alnemri et al., J.Biol. Chem. 269:30761-30764 (1994)). Cells were then examinedmicroscopically for morphological signs of apoptosis such as blebbing ofthe cytoplasmic membrane, condensation of nuclear chromatin and releaseof small apoptotic bodies. In addition the genomic DNA was examined forinternucleosomal DNA cleavage.

For the construction of transfer vectors and recombinant baculoviruses,the full length Mch4 in pBluescript KS+ was excised with Bam HI andEcoRI and subcloned into a Bam HI/EcoRI cut pVL1393 vector (Invitrogen,San Diego, Calif.) to generate the pVL-Mch4 transfer vector. ThepVL-CPP32 transfer vector was made as described previously(Fernandes-Alnemri et al., supra (1994)). The recombinant transfervectors were then used to generate recombinant Baculoviruses aspreviously described (Summers et al., "Manual of Methods for BaculovirusVectors and Insert Culture Procedures," Texas Experimental StationBulletin No. 1555 (Texas A&M University, College Station, Tex. (1987);and Alnemri et al., J. Biol. Chem. 266:3925-3936 (1991)).

For the induction of apoptosis in Sf9 cells by Mch4 and CPP32 cells wereinfected with recombinant baculoviruses AcNPV-Mch4 or AcNPV-CPP32.Apoptosis was measured microscopically by counting cells with theappropriate morphology (blebbing, nuclear condensation). Alternatively,internucleosomal DNA cleavage is assessed as a characteristic marker.Briefly, total cellular DNA is isolated at 42 h postinfection fromeither control Sf9 cells or Sf9 cells infected with AcNPV-Mch4 orAcNPV-CPP32 baculoviruses (Alnemri et al. supra (1995)). The DNA sampleswere analyzed by electrophoresis in a 1.8% agarose gel containingethidium bromide.

Expression of full length Mch4 in Sf9 cells caused a significantpercentage of the cells to undergo apoptosis by about 48 h postinfectionwhich is also manifested by induction of internucleosomal DNA cleavage.These results are consistent with Mch4 being a cell death protease sinceAcNPV-CPP32 yielded similar results.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 63                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3535 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 546..1584                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..3535                                                         (D) OTHER INFORMATION: /note= "Mch4"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TGAAGTCTCTTCCCAAGCAAATGGGAGCTTCTTTGGACCTTGGAGCACACAGAGGATTCT60                ACTTTCTTTAAAACTTTGTTTTCAGGCAATTTCCCTGAGAACCGTTTACTTCCAGAAGAT120               TGGTGGAGCTTGATCTGAAGGCTGGCCATGAAATCTCAAGGTCAACATTGGTATTCCAGT180               TCAGATAAAAACTGTAAAGTGAGCTTTCGTGAGAAGCTTCTGATTATTGATTCAAACCTG240               GGGGTCCAAGATGTGGAGAACCTCAAGTTTCTCTGCATAGGATTGGTCCCCAACAAGAAG300               CTGGAGAAGTCCAGCTCAGCCTCAGATGTTTTTGAACATCTCTTGGCAGAGGATCTGCTG360               AGTGAGGAAGACCTTTCTTCCTGGCAGAACTCCTCTATATCATACGGCAGAAGAAGCTGC420               TGCAGCACCTCAACTGTACCAAAGAGGAAGTGGAGCGACTGCTGCCCACCCGACAAAGGG480               TTTCTCTGTTTAGAAACCTGCTCTACGAACTGTCAGAAGGCATTGACTCAGAGAACTTAA540               AGGACATGATCTTCCTTCTGAAAGACTCGCTTCCCAAAACTGAAATG587                            MetIlePheLeuLeuLysAspSerLeuProLysThrGluMet                                    1510                                                                          ACCTCCCTAAGTTTCCTGGCATTTCTAGAGAAACAAGGTAAAATAGAT635                           ThrSerLeuSerPheLeuAlaPheLeuGluLysGlnGlyLysIleAsp                              15202530                                                                      GAAGATAATCTGACATGCCTGGAGGACCTCTGCAAAACAGTTGTACCT683                           GluAspAsnLeuThrCysLeuGluAspLeuCysLysThrValValPro                              354045                                                                        AAACTTTTGAGAAACATAGAGAAATACAAAAGAGAGAAAGCTATCCAG731                           LysLeuLeuArgAsnIleGluLysTyrLysArgGluLysAlaIleGln                              505560                                                                        ATAGTGACACCTCCTGTAGACAAGGAAGCCGAGTCGTATCAAGGAGAG779                           IleValThrProProValAspLysGluAlaGluSerTyrGlnGlyGlu                              657075                                                                        GAAGAACTAGTTTCCCAAACAGATGTTAAGACATTCTTGGAAGCCTTA827                           GluGluLeuValSerGlnThrAspValLysThrPheLeuGluAlaLeu                              808590                                                                        CCGAGGGCAGCTGTGTACAGGATGAATCGGAACCACAGAGGCCTCTGT875                           ProArgAlaAlaValTyrArgMetAsnArgAsnHisArgGlyLeuCys                              95100105110                                                                   GTCATTGTCAACAACCACAGCTTTACCTCCCTGAAGGACAGACAAGGA923                           ValIleValAsnAsnHisSerPheThrSerLeuLysAspArgGlnGly                              115120125                                                                     ACCCATAAAGATGCTGAGATCCTGAGTCATGTGTTCCAGTGGCTTGGG971                           ThrHisLysAspAlaGluIleLeuSerHisValPheGlnTrpLeuGly                              130135140                                                                     TTCACAGTGCATATACACAATAATGTGACGAAAGTGGAAATGGAGATG1019                          PheThrValHisIleHisAsnAsnValThrLysValGluMetGluMet                              145150155                                                                     GTCCTGCAGAAGCAGAAGTGCAATCCAGCCCATGCCGACGGGGACTGC1067                          ValLeuGlnLysGlnLysCysAsnProAlaHisAlaAspGlyAspCys                              160165170                                                                     TTCGTGTTCTGTATTCTGACCCATGGGAGATTTGGAGCTGTCTACTCT1115                          PheValPheCysIleLeuThrHisGlyArgPheGlyAlaValTyrSer                              175180185190                                                                  TCGGATGAGGCCCTCATTCCCATTCGGGAGATCATGTCTCACTTCACA1163                          SerAspGluAlaLeuIleProIleArgGluIleMetSerHisPheThr                              195200205                                                                     GCCCTGCAGTGCCCTAGACTGGCTGAAAAACCTAAACTCTTTTTCATC1211                          AlaLeuGlnCysProArgLeuAlaGluLysProLysLeuPhePheIle                              210215220                                                                     CAGGCCTGCCAAGGTGAAGAGATACAGCCTTCCGTATCCATCGAAGCA1259                          GlnAlaCysGlnGlyGluGluIleGlnProSerValSerIleGluAla                              225230235                                                                     GATGCTCTGAACCCTGAGCAGGCACCCACTTCCCTGCAGGACAGTATT1307                          AspAlaLeuAsnProGluGlnAlaProThrSerLeuGlnAspSerIle                              240245250                                                                     CCTGCCGAGGCTGACTTCCTACTTGGTCTGGCCACTGTCCCAGGCTAT1355                          ProAlaGluAlaAspPheLeuLeuGlyLeuAlaThrValProGlyTyr                              255260265270                                                                  GTATCCTTTCGGCATGTGGAGGAAGGCAGCTGGTATATTCAGTCTCTG1403                          ValSerPheArgHisValGluGluGlySerTrpTyrIleGlnSerLeu                              275280285                                                                     TGTAATCATCTGAAGAAATTGGTCCCAAGACATGAAGACATCTTATCC1451                          CysAsnHisLeuLysLysLeuValProArgHisGluAspIleLeuSer                              290295300                                                                     ATCCTCACTGCTGTCAACGATGATGTGAGTCGAAGAGTGGACAAACAG1499                          IleLeuThrAlaValAsnAspAspValSerArgArgValAspLysGln                              305310315                                                                     GGAACAAAGAAACAGATGCCCCAGCCTGCTTTCACACTAAGGAAAAAA1547                          GlyThrLysLysGlnMetProGlnProAlaPheThrLeuArgLysLys                              320325330                                                                     CTAGTATTCCCTGTGCCCCTGGATGCACTTTCAATATAGCAGAGAGT1594                           LeuValPheProValProLeuAspAlaLeuSerIle                                          335340345                                                                     TTTTGNTGGTTCTTAGACCTCAAACGAATCATTGGNTATAACCTCCAGCCTCCTGCCCAG1654              CACAGGAATCGGTGGTCTCCACCTGTCATTCTAGAAACAGGAAACACCGTGTTTTCTGAC1714              ACAGTCAATTCTGATTTTCTTTTTCTTTTGCAAGTCTAAATGTTAGAAAACTTTCTTTTT1774              TTGGAGATAGTCTCATTCTGTCACCCAGACTGGAGTGCAGGGGGGCAATCACGGCTCACT1834              GTAGTCTCGACCTCCCAGGCTCAAGCTGTCCTCCCACCTCAGCCTCCCAAGTAGCTGAGA1894              CTACAGGTGTGTGTCCATGCACAGCTAACTTTTTATTTTTTTTGNGGAGATGGGGTTTCA1954              CTATGTTGCCTAAGCTGGTCTCAAACTCCTGGGNTCAAGCGATCCTCCCACCTCAGCTTC2014              TCAAAGTTCTGGGACTACAGGCATGAAATACTGTGCCTGGCCTGGGGACCAGGTGCATTT2074              TAAGGTTCCTTGGTGTTCAAAAACCACGTTCTTAGCCTAGATTGAGCTTAGATTGCCTCT2134              CTAGACAACTACCCCTTAGTTATAATTCTGTGTCCCCTCTGCATGCCCTTAAACATTGGA2194              CAGTGAGGTCACAGTCCACCCACCCTCTCTCTGATCTCCCCCTTCCTAAGACTTCTCTTT2254              TGCACATCTAGTGAGGTGAAAATTTGGTCTATGCCAGGCCCATTTCCTGCTTTTGTGTAA2314              GGAAGGTGCTCACATAGGAAGTTTTTATTTGGTTAGAGACAGGTTTCCCTGTAGGAAGAT2374              GATGGCTCATTTACACTCAGCTGCTCTGCAAGCAGAAACTTTACAACCTGATGTCATATT2434              CCATTTTGGACTGGGTGCGGTGACTCATGCCTGTAATCCCAGTACTCTGGGAAGCCAAGG2494              CAGGCAGATCACTTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAATACGGCAAAACCTCA2554              TCATTACTAAAAACACAAAAATTAGCCAGGTGTGGCGGCGAGCACCTGTAATCCCAGCTA2614              CTCGGGAGGCTGAGACAGGAGAATCTCTTGAATCCAGGAGGCAGAGGCTGTGGTGAGCCA2674              AGATGACACAACTGCACTCCAGCTTGGGCAACAGGGCGAGACCTTGTTTAAAAAAAAAAT2734              TCAATATTGGGGTTGGAACATTTCAGTTGCCATTGACAGAACACCCAATTCAAATTGACT2794              GAAGCAAAGAAGGGAATTTATTGCCTCTTTCACATTGAAACCCAGGAGTGGATAACACTG2854              GCTTCAGGCAAAGCTTGAATCAGGACTCAATCTACAGGCCAGCACCTTTCTCTTGGCCGG2914              ATGTCCTCAGGGCTGGCAGATGCAGTAGACTGCAGTGGACAGTCCCCACCTTGTTACTGC2974              TACTACACTTTGCTCCTCTGGCCCAAGGCATGAGGAGAGAGGCTGTGTCAGAAACTGAAG3034              CTGTTCTCAGGATCACTGGGCTCTTCTTGGCAGAGGGGATGTCTGGCTTGCCTGAAGGGA3094              GTGGCTCTGTAAGGACGCCTTGATGCTTTCTTCATTAAGATTTTGAGCATTTTTACGTAC3154              TTGAGCTTTTTTTTTTTTTTTTTTCAATTTCTAGAGGAACTTTTTCTCTGTTAATTCCTG3214              GAACTGTATTTTGAATCCTTAAAGGTGAGCCCTCATAGGGAGATCCAAAGTCCTGTGGTT3274              AACGCCTTCATTTATAGATGAGGCAGCTGAGGCCTGGGGATGTGAACAACCTGCTCACAG3334              TCCTCATTTACTGGATTTGACTTCAGCCAGGTGAACTGGAATGCCTTGGGGCGTGGAAGG3394              GCATTAGGAGTGTTTCATTTGATATGTGAATGCTCATAAAAAAATGTCAAGGAATGAAGA3454              ACAACAACTCTCAGTGGTGCCTGCATTTATAATTATTTATGTGAAAGTCAAATTCATGTA3514              CAGTAAATTTGTTATAAGAAT3535                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 346 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetIlePheLeuLeuLysAspSerLeuProLysThrGluMetThrSer                              151015                                                                        LeuSerPheLeuAlaPheLeuGluLysGlnGlyLysIleAspGluAsp                              202530                                                                        AsnLeuThrCysLeuGluAspLeuCysLysThrValValProLysLeu                              354045                                                                        LeuArgAsnIleGluLysTyrLysArgGluLysAlaIleGlnIleVal                              505560                                                                        ThrProProValAspLysGluAlaGluSerTyrGlnGlyGluGluGlu                              65707580                                                                      LeuValSerGlnThrAspValLysThrPheLeuGluAlaLeuProArg                              859095                                                                        AlaAlaValTyrArgMetAsnArgAsnHisArgGlyLeuCysValIle                              100105110                                                                     ValAsnAsnHisSerPheThrSerLeuLysAspArgGlnGlyThrHis                              115120125                                                                     LysAspAlaGluIleLeuSerHisValPheGlnTrpLeuGlyPheThr                              130135140                                                                     ValHisIleHisAsnAsnValThrLysValGluMetGluMetValLeu                              145150155160                                                                  GlnLysGlnLysCysAsnProAlaHisAlaAspGlyAspCysPheVal                              165170175                                                                     PheCysIleLeuThrHisGlyArgPheGlyAlaValTyrSerSerAsp                              180185190                                                                     GluAlaLeuIleProIleArgGluIleMetSerHisPheThrAlaLeu                              195200205                                                                     GlnCysProArgLeuAlaGluLysProLysLeuPhePheIleGlnAla                              210215220                                                                     CysGlnGlyGluGluIleGlnProSerValSerIleGluAlaAspAla                              225230235240                                                                  LeuAsnProGluGlnAlaProThrSerLeuGlnAspSerIleProAla                              245250255                                                                     GluAlaAspPheLeuLeuGlyLeuAlaThrValProGlyTyrValSer                              260265270                                                                     PheArgHisValGluGluGlySerTrpTyrIleGlnSerLeuCysAsn                              275280285                                                                     HisLeuLysLysLeuValProArgHisGluAspIleLeuSerIleLeu                              290295300                                                                     ThrAlaValAsnAspAspValSerArgArgValAspLysGlnGlyThr                              305310315320                                                                  LysLysGlnMetProGlnProAlaPheThrLeuArgLysLysLeuVal                              325330335                                                                     PheProValProLeuAspAlaLeuSerIle                                                340345                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1355 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 50..1217                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..1355                                                         (D) OTHER INFORMATION: /note= "Mch5"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GAATTCGGCACGAGCTCAAATTTCTGCCTACAGGTTCCACTTCTGCCGCATGAGC55                     MetSer                                                                        TGGGCTGAAGCAAACAGCCAGTGCCAGACACAGTCTGTACCTTTCTGG103                           TrpAlaGluAlaAsnSerGlnCysGlnThrGlnSerValProPheTrp                              51015                                                                         CGGAGGGTCGATCATCTATTAATAAGGGTCATGCTCTATCAGATTTCA151                           ArgArgValAspHisLeuLeuIleArgValMetLeuTyrGlnIleSer                              202530                                                                        GAAGAAGTGAGCAGATCAGAATTGAGGTCTTTTAAGTTTCTTTTGCAA199                           GluGluValSerArgSerGluLeuArgSerPheLysPheLeuLeuGln                              35404550                                                                      GAGGAAATCTCCAAATGCAAACTGGATGATGACATGAACCTGCTGGAT247                           GluGluIleSerLysCysLysLeuAspAspAspMetAsnLeuLeuAsp                              556065                                                                        ATTTTCATAGAGATGGAGAAGAGGGTCATCCTGGGAGAAGGAAAGTTG295                           IlePheIleGluMetGluLysArgValIleLeuGlyGluGlyLysLeu                              707580                                                                        GACATCCTGAAAAGAGTCTGTGCCCAAATCAACAAGAGCCTGCTGAAG343                           AspIleLeuLysArgValCysAlaGlnIleAsnLysSerLeuLeuLys                              859095                                                                        ATAATCAACGACTATGAAGAATTCAGCAAAGGGGAGGAGTTGTGTGGG391                           IleIleAsnAspTyrGluGluPheSerLysGlyGluGluLeuCysGly                              100105110                                                                     GTAATGACGATGTCGGACTGTCCAAGAGAACAGGATAGTGAATCACAG439                           ValMetThrMetSerAspCysProArgGluGlnAspSerGluSerGln                              115120125130                                                                  ACTTTGGACAAAGTTTACCAAATGAAAAGCAAGCCTCGGGGATACTGT487                           ThrLeuAspLysValTyrGlnMetLysSerLysProArgGlyTyrCys                              135140145                                                                     CTGATCATCAACAATCACAATTTTGCAAAAGCACGGGAGAAAGTGCCC535                           LeuIleIleAsnAsnHisAsnPheAlaLysAlaArgGluLysValPro                              150155160                                                                     AAACTTCACAGCATTAGGGACAGGAATGGAACACACTTGGATGCAGGG583                           LysLeuHisSerIleArgAspArgAsnGlyThrHisLeuAspAlaGly                              165170175                                                                     GCTTTGACCACGACCTTTGAAGAGCTTCATTTTGAGATCAAGCCCCAC631                           AlaLeuThrThrThrPheGluGluLeuHisPheGluIleLysProHis                              180185190                                                                     CATGACTGCACAGTAGAGCAAATCTATGAGATTTTGAAAATCTACCAA679                           HisAspCysThrValGluGlnIleTyrGluIleLeuLysIleTyrGln                              195200205210                                                                  CTCATGGACCACAGTAACATGGACTGCTTCATCTGCTGTATCCTCTCC727                           LeuMetAspHisSerAsnMetAspCysPheIleCysCysIleLeuSer                              215220225                                                                     CATGGAGACAAGGGCATCATCTATGGCACTGATGGACAGGAGGCCCCC775                           HisGlyAspLysGlyIleIleTyrGlyThrAspGlyGlnGluAlaPro                              230235240                                                                     ATCTATGAGCTGACATCTCAGTTCACTGGTTTGAAGTGCCCTTCCCTT823                           IleTyrGluLeuThrSerGlnPheThrGlyLeuLysCysProSerLeu                              245250255                                                                     GCTGGAAAACCCAAAGTGTTTTTTATTCAGGCTTGTCAGGGGGATAAC871                           AlaGlyLysProLysValPhePheIleGlnAlaCysGlnGlyAspAsn                              260265270                                                                     TACCAGAAAGGTATACCTGTTGAGACTGATTCAGAGGAGCAACCCTAT919                           TyrGlnLysGlyIleProValGluThrAspSerGluGluGlnProTyr                              275280285290                                                                  TTAGAAATGGATTTATCATCACCTCAAACGAGATATATCCCGGATGAG967                           LeuGluMetAspLeuSerSerProGlnThrArgTyrIleProAspGlu                              295300305                                                                     GCTGACTTTCTGCTGGGGATGGCCACTGTGAATAACTGTGTTTCCTAC1015                          AlaAspPheLeuLeuGlyMetAlaThrValAsnAsnCysValSerTyr                              310315320                                                                     CGAAACCCTGCAGAGGGAACCTGGTACATCCAGTCACTTTGCCAGAGC1063                          ArgAsnProAlaGluGlyThrTrpTyrIleGlnSerLeuCysGlnSer                              325330335                                                                     CTGAGAGAGCGATGTCCTCGAGGCGATGATATTCTCACCATCCTGACT1111                          LeuArgGluArgCysProArgGlyAspAspIleLeuThrIleLeuThr                              340345350                                                                     GAAGTGAACTATGAAGTAAGCAACAAGGATGACAAGAAAAACATGGGG1159                          GluValAsnTyrGluValSerAsnLysAspAspLysLysAsnMetGly                              355360365370                                                                  AAACAGATGCCTCAGCCTACTTTCACACTAAGAAAAAAACTTGTCTTC1207                          LysGlnMetProGlnProThrPheThrLeuArgLysLysLeuValPhe                              375380385                                                                     CCTTCTGATTGATGGTGCTATTTTGTTTGTTTTGTTTTGTTTTGTTTTTT1257                        ProSerAsp                                                                     TGAGACAGAATCTCGCTCTGTCGCCCAGGCTGGAGTGCAGTGGCGTGATCTCGGCTCACC1317              GCAAGCTCCGCCTCCCGGGTTCAGGCCATTCTCCTGCT1355                                    (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 389 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetSerTrpAlaGluAlaAsnSerGlnCysGlnThrGlnSerValPro                              151015                                                                        PheTrpArgArgValAspHisLeuLeuIleArgValMetLeuTyrGln                              202530                                                                        IleSerGluGluValSerArgSerGluLeuArgSerPheLysPheLeu                              354045                                                                        LeuGlnGluGluIleSerLysCysLysLeuAspAspAspMetAsnLeu                              505560                                                                        LeuAspIlePheIleGluMetGluLysArgValIleLeuGlyGluGly                              65707580                                                                      LysLeuAspIleLeuLysArgValCysAlaGlnIleAsnLysSerLeu                              859095                                                                        LeuLysIleIleAsnAspTyrGluGluPheSerLysGlyGluGluLeu                              100105110                                                                     CysGlyValMetThrMetSerAspCysProArgGluGlnAspSerGlu                              115120125                                                                     SerGlnThrLeuAspLysValTyrGlnMetLysSerLysProArgGly                              130135140                                                                     TyrCysLeuIleIleAsnAsnHisAsnPheAlaLysAlaArgGluLys                              145150155160                                                                  ValProLysLeuHisSerIleArgAspArgAsnGlyThrHisLeuAsp                              165170175                                                                     AlaGlyAlaLeuThrThrThrPheGluGluLeuHisPheGluIleLys                              180185190                                                                     ProHisHisAspCysThrValGluGlnIleTyrGluIleLeuLysIle                              195200205                                                                     TyrGlnLeuMetAspHisSerAsnMetAspCysPheIleCysCysIle                              210215220                                                                     LeuSerHisGlyAspLysGlyIleIleTyrGlyThrAspGlyGlnGlu                              225230235240                                                                  AlaProIleTyrGluLeuThrSerGlnPheThrGlyLeuLysCysPro                              245250255                                                                     SerLeuAlaGlyLysProLysValPhePheIleGlnAlaCysGlnGly                              260265270                                                                     AspAsnTyrGlnLysGlyIleProValGluThrAspSerGluGluGln                              275280285                                                                     ProTyrLeuGluMetAspLeuSerSerProGlnThrArgTyrIlePro                              290295300                                                                     AspGluAlaAspPheLeuLeuGlyMetAlaThrValAsnAsnCysVal                              305310315320                                                                  SerTyrArgAsnProAlaGluGlyThrTrpTyrIleGlnSerLeuCys                              325330335                                                                     GlnSerLeuArgGluArgCysProArgGlyAspAspIleLeuThrIle                              340345350                                                                     LeuThrGluValAsnTyrGluValSerAsnLysAspAspLysLysAsn                              355360365                                                                     MetGlyLysGlnMetProGlnProThrPheThrLeuArgLysLysLeu                              370375380                                                                     ValPheProSerAsp                                                               385                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..19                                                           (D) OTHER INFORMATION: /note= "t96-pr1"                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TCAGCCTCGGCAGGAATAC19                                                         (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..17                                                           (D) OTHER INFORMATION: /note= "SK-Zap"                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CAGGAATTCGGCACGAG17                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: /note= "Mch5-pr1"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GACAGAGCGAGATTCTGT18                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: /note= "Mch5-pr2"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GCACCATCAATCAGAAGG18                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: /note= "t96-pr5"                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CGGGAGATCATGTCTCAC18                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GlnAlaCysGlnGly                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GlnAlaCysArgGly                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "Mch5"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ArgAspArgAsnGlyThr                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "Mch5"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      LeuSerHisGlyAspLys                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION: /note= "Mch5"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      PheIleGlnAlaCysGlnGlyAspAsn                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "Mch5"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ValGluThrAspSer                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: /note= "Mch5"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AsnCysValSerTyrArgAsnProAlaGluGlyThrTrpTyrIle                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "Mch4"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      LysAspArgGlnGlyThr                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "Mch4"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      LeuThrHisGlyArgPhe                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION: /note= "Mch4"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      PheIleGlnAlaCysGlnGlyGluGlu                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "Mch4"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      IleGluAlaAspAla                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: /note= "Mch4"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GlyTyrValSerPheArgHisValGluGluGlySerTrpTyrIle                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "Mch3"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GlyValArgAsnGlyThr                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "Mch3"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      LeuSerHisGlyGluGlu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION: /note= "Mch3"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      PheIleGlnAlaCysArgGlyThrGlu                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "Mch3"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      IleGlnAlaAspSer                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: /note= "Mch3"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      GlyTyrTyrSerTrpArgSerProGlyArgGlySerTrpPheVal                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "Mch2"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      ProGluArgArgGlyThr                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "Mch2"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      LeuSerHisGlyGluGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION: /note= "Mch2"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      IleIleGlnAlaCysArgGlyAsnGln                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "Mch2"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      ThrGluValAspAla                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: /note= "Mch2"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      GlyTyrTyrSerHisArgGluThrValAsnGlySerTrpTyrIle                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "CPP32"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      ThrSerArgSerGlyThr                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "CPP32"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      LeuSerHisGlyGluGlu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION: /note= "CPP32"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      IleIleGlnAlaCysArgGlyThrGlu                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "CPP32"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      IleGluThrAspSer                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: /note= "CPP32"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      GlyTyrTyrSerTrpArgAsnSerLysAspGlySerTrpPheIle                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "CED-3"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      ProThrArgAsnGlyThr                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "CED-3"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      LeuSerHisGlyGluGlu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION: /note= "CED-3"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      PheValGlnAlaCysArgGlyGluArg                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "CED-3"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      AspSerValAspGly                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: /note= "CED-3"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      GlnTyrValSerTrpArgAsnSerAlaArgGlySerTrpPheIle                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "ICE"                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      ProArgArgThrGlyAla                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "ICE"                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      MetSerHisGlyIleArg                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION: /note= "ICE"                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      IleIleGlnAlaCysArgGlyAspSer                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "ICE"                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      TrpPheLysAspSer                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: /note= "ICE"                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      AspAsnValSerTrpArgHisProThrMetGlySerValPheIle                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "TX"                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      ProProArgAsnGlyAla                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "TX"                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      MetSerHisGlyIleLeu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION: /note= "TX"                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      IleValGlnAlaCysArgGlyAlaAsn                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "TX"                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      TrpValLysAspSer                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: /note= "TX"                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      HisAsnValSerTrpArgAspSerThrMetGlySerIlePheIle                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "ICErelIII"                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      ProAlaArgAsnGlyAla                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "ICErelIII"                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      MetSerHisGlyIleLeu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION: /note= "ICErelIII"                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      IleValGlnAlaCysArgGlyGluLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "ICErelIII"                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      TrpValArgAspSer                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: /note= "ICErelIII"                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      HisAsnValSerTrpArgAspArgThrArgGlySerIlePheIle                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "ICH-1"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      GluPheArgSerGlyGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: /note= "ICH-1"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      LeuSerHisGlyValGlu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION: /note= "ICH-1"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      PheIleGlnAlaCysArgGlyAspGlu                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /note= "ICH-1"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      AspGlnGlnAspGly                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: /note= "ICH-1"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      GlyThrAlaAlaMetArgAsnThrLysArgGlySerTrpTyrIle                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      GlySerTrpPheIle                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      GlySerTrpTyrIle                                                               15                                                                            __________________________________________________________________________

What is claimed is:
 1. An isolated Mch4 polypeptide comprising an aminoacid sequence selected from the group consisting of:SEQ ID NO:2; aminoacids 1 through 239 of SEQ ID NO:2; amino acids 102 through 346 of SEQID NO:2; and amino acids 102 through 239 of SEQ ID NO:2.
 2. The isolatedMch4 polypeptide of claim 1, wherein said amino acid sequence is SEQ IDNO:2.
 3. The isolated Mch4 polypeptide of claim 1, wherein said aminoacid sequence is amino acids 1 through 239 of SEQ ID NO:2.
 4. Theisolated Mch4 polypeptide of claim 1, wherein said amino acid sequenceis amino acids 102 through 346 of SEQ ID NO:2.
 5. The isolated Mch4polypeptide of claim 1, wherein said amino acid sequence is amino acids102 through 239 of SEQ ID NO:2.
 6. An isolated Mch5 polypeptidecomprising an amino acid sequence selected from the group consistingof:SEQ ID NO:4; and amino acids 1 through 284 of SEQ ID NO:4.
 7. Theisolated Mch5 polypeptide of claim 6, wherein said amino acid sequenceis SEQ ID NO:4.
 8. The isolated Mch5 polypeptide of claim 6, whereinsaid amino acid sequence is amino acids 1 through 284 of SEQ ID NO:4.